Triple Epitope Tag Sequences for Antibody Fragment Detection
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Solution Overview
Problem
Existing epitope tag combinations are often inaccessible due to protein secondary and tertiary structures, and can alter protein expression and stability in display systems, limiting detection, capture, and purification of antibody fragments like single chain antibodies and Fab fragments.
Innovation Solution
A novel triple tag sequence comprising a 6X histidine tag, a c-myc tag positioned between the 6X histidine and V5 tag, which remains accessible for antibody binding, allowing for efficient detection, capture, and purification of proteins of interest without affecting their expression or display in libraries.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If epitope tags are inserted into proteins for detection and purification, then detection and purification capability is improved, but tag accessibility is reduced due to protein secondary and tertiary structures
Solution Approach 1:
The invention divides the tagging function into multiple separate epitope tags (V5, c-myc, 6X histidine) positioned at different locations on the protein. This segmentation ensures that at least one tag remains accessible for detection and purification even if others are buried in protein structure, resolving the contradiction between detection capability and tag accessibility.
2Reliability
If multiple epitope tags are added to proteins for enhanced detection, then detection capability is improved, but protein expression and stability are altered
Solution Approach 1:
The invention applies different types of epitope tags (V5, c-myc, 6X histidine) with distinct properties at different positions on the protein. Each tag has been selected for its specific binding characteristics and minimal interference with protein folding and stability, allowing enhanced detection capability while maintaining protein expression and stability.
3Productivity
If antibody fragments like scFv and Fab are captured using Fc region reagents, then capture efficiency is improved, but capture is precluded for fragments lacking Fc domains
Solution Approach 1:
The invention creates a universal tagging system that can be applied to all antibody fragments regardless of their class or presence of Fc domains. The multiple epitope tags (V5, c-myc, 6X histidine) provide universal capture handles that work with scFv, Fab, and other fragments lacking Fc regions, greatly expanding the versatility of capture methods while maintaining efficiency.
4Reliability
If Fab fragments are captured using anti-Fab antibodies, then capture capability is improved, but antigen binding is interfered with
Solution Approach 1:
The invention extracts the capture function from the antibody-antigen binding site by using separate epitope tags (V5, c-myc, 6X histidine) that are distinct from the antigen-binding Fab region. This allows capture of Fab fragments using anti-tag antibodies without interfering with the intact antigen-binding capability of the Fab region.
Data Source
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AI summary
The present disclosure relates to novel triple tag sequences that may comprise a 6X histidine tag, a c-myc tag and a V5 tag. The present disclosure also provides polynucleotides, proteins, vectors and host cells that comprise the triple tag sequence of the present disclosure, including libraries of such polynucleotides, proteins, vectors and host cells. The novel triple tag sequences of the present disclosure may be used in phage display vectors and phage libraries and in methods for detection, screening, capture, purification, quantitation, and/or recovery of proteins of interest to which they are linked. Proteins of interest include antibodies such as single chain antibodies, single chain antibodies, and Fab fragments of antibodies or peptides such as non-antibody peptides.